Utilization of stem cells to repair diseased or damaged tissues/organs represents a novel approach for remedies in regenerative medicine. Although embryonic stem cells (ESC) and induced pluripotent stem cells (iPS) have been studied, there are major drawbacks and concerns in using ESC and iPS; for example, tumorigenesis and teratomas formation can occur after their transplantation. It is not clear if the obstacles pertaining to ESC and iPS can be overcome in order to use them for therapeutics; at least there is still long way to go. In contrast, adult stem cells (AdSC) are used for treatment of human diseases. One example is the transplantation of bone marrow stem cells to cure or improve certain disorders, such as leukemia. Such cell- based therapies usually require large numbers of stem cells. Because AdSC lose their stem cell properties (stemness) including differentiation capability when cultured in vitro, primary isolated AdSC can only be used for limited passages, and propagation of a large quantity of high-potential stem cells from primary isolation is difficult. Thus, large amounts of tissues are needed for stem cell isolation, and continuous isolation of AdSC is required for multiple treatments. However, tissues, e.g., dental tissues that can be used for AdSC isolation are often limited. Stem cells isolated from dental pulp of extracted and exfoliated teeth have been shown to be a good source of stem cells for regenerative medicine. However, like other AdSC, these dental pulp stem cells (DPSCs) also lose their differentiation potential when cultured in vitro, which hampers their applications. The long-term goal of this research is to elucidate the genes and molecules regulating differentiation capability in the DPSCs. Preliminary studies showed that heat shock protein B8 (HspB8) was downregulated in cultured DPSCs when the cells lost differentiation capability, and knockdown of HspB8 expression in the early passage DPSCs caused the cells to lose differentiation capability. Further studies suggested that microRNAs (miRNAs) are responsible for downregulation of HspB8 expression in DPSCs. The central hypothesis is that upregulated expression of certain miRNAs in late passage DPSCs downregulates HspB8 and other regulatory genes, and in turn causes DPSCs to lose differentiation capability during in vitro culture. The project Aims: (1) to identify the miRNAs whose expression is substantially increased in late passage DPSCs, designated as late-passage upregulated miRNAs (LPU-miRNAs); (2) to identify the LPU-miRNAs that downregulate HspB8 expression; i.e., HspB8-targeting miRNAs, and (3) to study the effects of LPU-miRNAs and HspB8-targeting miRNAs on DPSC differentiation and on regulating other potential genes. Accomplishment of the Aims would be significant in understanding the roles of miRNAs and HspB8 in regulating the loss of differentiation seen in the long-term cultured DPSCs and will shed light on other AdSC as well. This would facilitate the development of cutting- edge methods for maintaining the stem cell properties in culturing DPSCs and AdSC, such that a large quantity of stem cells with high-differentiation potential can be obtained from expansion of primary isolated stem cells.